One key to the development of successful treatment for solid tumors is the ability to selectively and specifically kill solid tumor cells, while leaving healthy, non-tumor cells intact. Unfortunately, there are few measurable qualitative differences between neoplastic cells present in solid tumors and the healthy cells of normal tissue. Thus, treating a solid tumor inevitably results in harmful non-specific effects in a subject (e.g., neuronal toxicity). Although treatments like chemotherapy and radiation therapy have been successful for targeting solid tumors, many of the most prevalent types are resistant to these methods.
For example, malignant gliomas, the largest group of primary intracranial brain tumors, represent a therapeutic problem which is not solved yet. Although the knowledge of the biology of these tumors has grown due to intense basic research, clinical progress and prognosis are still very poor.
Malignant gliomas are tumors of neuroepithelial origin and are cytologically divided in ependimomas, oligodendrogliomas, oligoastrocytomas, astrocytomas and glioblastomas. With a proportion of more than 60%, diffuse-infiltrating astrocytomas (WHO Grade II-IV) represent the largest group of intracranial tumors. The WHO classification revised in 2001 is largely established as grading scheme for astrocytomas. According to the WHO, brain tumors are allocated by means of histological criteria to 4 malignancy grades (Kleihus and Cavenee, 2000). The prognosis for diffuse-infiltrating astrocytomas is generally poor. The prognosis depends, on the one hand, on the malignancy grade and, on the other, on the localization of the tumor and the therapy procedure. The average survival rate for patients with an astrocytoma WHO grade II is more than 5 years, with an astrocytoma WHO grade III, 2 to 5 years, and with an astrocytoma-glioblastoma of the WHO grade IV (=glioblastoma), less than 1 year.
The molecular pathogenesis of tumors is a complex process and is based on mutations of different genes which are responsible for the control of the cell cycle. Mutations in the tumor suppressor gene p53 are the most frequently found alterations in human tumors and are also responsible for the development of low-grade astrocytomas as well as for the progression to the secondary glioblastoma. However, primary developed glioblastomas very rarely have p53 mutations. A further gene, which indicates a malignant tendency of diffuse astrocytomas, is suspected to be on the long arm of chromosome 19. Further genes which are frequently altered in case of glioblastomas are the oncogenes MDM2 and MDM4 and also the tumor suppressor gene p14ARF, which are involved in the p53-dependent control of the cell cycle (Dai and Holland, 2001). An amplification of the EGF receptor gene is observed in 30-40% of the primary glioblastomas and is therefore the most frequently amplified oncogene in this tumor group (Holland, 2001). The majority of malignant gliomas responds poorly to a chemo- or radiotherapy. It is assumed that the reason for this is mutations of cell-cycle-associated genes which are also involved in the regulation of the apoptosis.
More effective therapy methods for malignant gliomas are urgently needed because from the existing therapy methods such as chemo- or radiotherapy, no significant improvements for the prognosis of the disease are to be expected. In contrast, the gene therapy of the glioblastoma offers promising possibilities which need to be exploited. A plurality of different, very effective genes was developed for this purpose. For most of them, data from experiments on animals are available (Shir and Levitzki, 2001). These therapeutic genes can be allocated to four different active principles:
(i) The gene product of so-called suicide genes converts precursors in cytotoxic molecules. An example is the thymidine kinase (TK) of the herpes simplex virus (HSV) in connection with a dosage of ganciclovir. A particular advantage is that the toxic ganciclovir triphosphate diffuses into adjacent cells, whereby a bystander effect takes place. In the last years, the activity of this enzyme was further increased. HSV TK is currently a very efficient possibility to eliminate tumor cells as well as implanted vector producing cells.
(ii) The expression of immunostimulatory cytokines such as the IL-4 can stimulate the natural defense against tumor cells.
(iii) The secretion of anti-angiogenetic proteins such as the endostatin results in a lack of blood vessels and therefore in a lack of nutrient supply in the metabolically very active tumor tissue. The tumor is virtually “starving”.
(iv) Finally, a series of genes was described which engage into the signal transduction or the cell cycle of the tumor cell in order to inhibit the uncontrolled growth of these cells. However, the possibilities of use of these genes in the clinic are limited because these genes act only in the gene-modified cell itself and do not have the bystander effect as the first-mentioned active principles. This means that in order to achieve a therapeutic effect, virtually all malignant cells have to be genetically modified which is hopeless even with ideal vector systems.
One of the most important prerequisite for a successful gene therapy of the glioblastoma is provided by the multitude of existing, very effective principles of action. However, a problem which is not solved yet is the inefficient gene transfer and a poor expression of the therapeutic gene in the target cells. This is also the reason why, despite the multitude of efficient therapeutic genes, the gene therapy of glioblastoma failed in the clinic.
One advantage of the viral gene transfer over physicochemical transfection methods is the higher gene transfer rate and the long term expression of the genes because the viruses have developed particularly efficient mechanisms to introduce their genome into cells and to express it. In particular replication-competent viruses such as, amongst others, herpes simplex virus (HSV), adenoviruses (Ad), Newcastle Disease Virus (NDV) and the vesicular stomatitis virus (VSV) are currently used as oncolytic viruses (OV). For an optimal virotherapy of glioblastoma, the OV should have the following features:
(i) They should have a tumor-specific tropism, whereby virus replication and cell lysis remains limited to the tumor tissue. This property can be enhanced by modification of the viral envelope or by using tumor-specific promoters. Since the assumption is that only a small portion of the glioma cells divide during the treatment, viruses which infect resting cells as well as proliferating cells are of advantage.
(ii) With respect to safety-relevant aspects, viruses with high genetic stability and a low toxicity outside of the tumor tissue are particularly suitable for clinical use. This allows a high virus titer and a purification of the vectors under GMP conditions. Ideally, the OV should be apathogenic for humans and should have a low infection rate among the population. An already existing immunity would result in a premature neutralization of the virus and thus would not allow an efficient therapy.
Prominent examples for oncolytic viruses in the therapy of glioblastomas are the attenuated HSV variants G207 and 1716 and the adenovirus ONYX-015. One objection against the use of oncolytic HSV for the treatment of CNS tumors is its high level replication in normal brain cells which can result in a life-threatening encephalitis. Moreover, besides potential persistence, there is the possibility of reactivation of latent HSV. The HSV variant 1716, which was generated by deleting a plurality of genes, selectively replicates in rapidly proliferating cells of the CNS but not in postmitotic neurons. Thereby, the neurotoxicity of HSV was significantly reduced. The treatment of experimental gliomas in the rat and in the mouse with HSV 1716 resulted in selective destruction of tumor cells while surrounding brain tissue remained undamaged. ONYX-015 is a further oncolytic virus which was developed for the glioblastoma therapy. Through a deletion in the E1B gene, this adenovirus is intended to selectively lyse cells with defective p53.
Oncolytic HSV as well as ONYX-015 were already clinically tested for the treatment of gliomas. Both attenuated oncolytic viruses showed a sufficient safety in clinical phase I/II studies. Independently of whether the viruses were injected intratumorally or into the resection cavity, the treatment was well tolerated and no serious side effects were observed. However, the cytolytic effects were only of transient nature, always followed by a recurrence (Cutter et al., 2206). Since normally the proliferation rate of gliomas exceeds the amplification rate and thus the spreading wave of viruses, the destruction of gliomas only by oncolysis is questionable. In fact, for an efficient treatment, the combination of a plurality of active principles is required. By using suicide genes or immunomodulatory genes in OV, a synergistic effect was demonstrated in preclinical studies (Tyminski et al., 2005; Fukuhara et al., 2005).
Besides the aforementioned DNA viruses, oncolytic RNA viruses are also under development. VSV is an enveloped negative-strand virus, the host spectrum of which includes rodents and livestock. Infections of humans are rare and are mostly asymptomatic. Due to the very low seroprevalence among the population, an impairment of the therapy efficiency by VSV-neutralizing antibodies is not to be expected. The infection and the cytoplasmatic replication of VSV take place independently of the cell cycle so that actively dividing cells and resting cells are equally infected. The efficient and preferential lysis of neoplastic cells by VSV is related to the mostly defective interferon signaling pathway and the accompanying viral replication in these cells (Wollmann et al., 2007). It was also demonstrated that independent of the cellular immune response, tumor cells with defects in the genes Myc, Ras or p53 also support the reproduction of VSV (Barber, 2004). The tumor specificity of VSV was further optimized in the last years by the preparation of recombinant viruses. The main focus here is on variants with mutation in the M protein (ΔM51). This variant is not able to prevent the interferon response in healthy cells, whereby virus replication in such cells is suppressed. In tumor cells with defective IFN response, the virus can replicate and thus be selectively oncolytically active. Even after systemic application, VSVΔM51 showed a secure and efficient oncolysis of human gliomas in the mouse model (Lun et al., 2006).
The application of viral vectors directly into the brain requires a high selectivity for tumor cells and is only possible in relatively small volumes. Thus, an efficient gene transfer can only be achieved with highly concentrated vector preparations (>108/ml) which have a strongly developed tropism for glioma cells. In case of gamma-retroviral and lentiviral vectors, vector tropism and vector stability can be influenced by integrating a non-retroviral envelope protein. In many cases, the retroviral envelope protein is replaced with the more stable G-protein of VSV. A problem of these so-called pseudotyped vectors is that VSV-G is cell-toxic, namely in the producer cells as well as for the surrounding healthy tissue, which previously stood in the way of a widespread use of such VSV-G pseudotypes in the clinic.
The inefficient gene transfer in vivo and not the lack of therapeutically effective genes currently hinders successful gene therapy of the glioblastoma. The previously known vectors for gene therapy and oncolytic virotherapy of gliomas are not optimal for various reasons. The efficiency, specificity and safety of previous gene transfer methods are to be increased to such an extent that a therapeutically effective gene transfer in patients is possible.
Therefore, developing a highly potent oncolytic viral gene transfer system for the treatment of solid tumors is highly desirable.